日本血吸虫信号通路相关基因SjWD101的生物信息学分析及其克隆与表达  

Bioinformative analysis of the Schistosoma japonicum signal transduction-related gene SiWD101 and its cloning and expression

作　　者：刘玲[1] 陈守义[1,2] 余新炳[1] 赵宇[1] 黄灿[1] 胡旭初[1] 徐劲[1] 

机构地区：[1]中山大学中山医学院寄生虫学教研室,广州510080 [2]广州市疾病预防控制中心,广州510080

出　　处：《中国人兽共患病学报》2009年第1期4-8,共5页Chinese Journal of Zoonoses

基　　金：国家自然科学基金(30571633)

摘　　要：目的利用生物信息学方法分析日本血吸虫SjWD101的结构和功能特征,并利用基因工程技术进行克隆和表达,获得相应的重组蛋白。方法从日本血吸虫GenBank数据库中筛选WD家族蛋白中全长基因,用相关软件进行生物信息学预测。通过RT-PCR扩增出全长编码区序列,克隆到表达载体pET-28a(+)中,在大肠杆菌BL21中经IPTG诱导表达,表达产物用镍离子金属螯合剂亲和层析柱纯化并经十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和蛋白印迹(Western blotting)鉴定。结果SjWD101是一个全长1085bp的基因,编码区为66～950,编码294个氨基酸,软件分析表明其理论分子量和等电点分别是31819.7Da和6.44.亚细胞定位分析表明该蛋白是胞浆蛋白,理化性质较稳定。二级结构以反向平行的β折叠为主。含有6个WD40结构域,属于WD蛋白家族中的一员,具有GTP结合蛋白的结构特征。经PCR、双酶切和测序证实pET-28a(+)-SjWD101构建成功。SDS-PAGE和Western blotting证实重组菌成功表达出融合蛋白,经His亲和层析柱获得了纯化的重组蛋白,Western blotting证实WD101重组蛋白能被感染日本血吸虫的兔血清识别。结论日本血吸虫WD101经生物信息学预测为WD家族蛋白,可能在血吸虫信号转导通路方面有其一定作用,本研究获得了纯化的重组蛋白,为进一步研究其生物学功能奠定了基础。To analyze the structural and functional characteristics of the Schistosoma japonicum signal transduction-related gene SjWD101 by bioinformatics and to clone and express the recombinant protein produced by genetic engineering, the full-length gene a WD-repeat containing family protein from S. japonicum was screened from GenBank data base and the characteristics of one member of the WD101 protein was predicted using related bioinformative softwares. The coding region was amplified by RT-PCR and then cloned into prokaryotic expression vector pET-28a（＋） and expressed in E. coli BL21 with IPTG induction. The expressed protein was purified by Ni-IDA affinity chromato-graphy and detected by SDS-PAGE and Western blotting. It was demonstrated that the SjWD101 gene was found to be 1085 bp in length with 66-950 coding regions which coded 294 amino acids and it was a complete full-length gene compared with the homologue in GenBank. Its theoretical molecular weight and isoelectric point were 31819.7 daltons and 6.44 respectively. As demonstrated by subcetlular analysis, the protein SjWD101 was a cytoplasmic protein with stable physical-chemical properties and it was mainly constructed with β-propellcr primary second structure, belonging to the member of the WD family ,which contained 6 WD40 domains. In addition, it had the structural characteristics of GTP-binding protein. The recombinant expression plasmid pET-28a（＋）-SjWD101 could be constructed successively after PCR, double enzyme digestion and DNA sequencing, and the recombinant fusion protein could be expressed in E. coil BL21 as demonstrated by SDS-PAGE and Western blotting. Meanwb.ile, the recombinant protein purified by Ni-IDA affinity chromatography could react specifically with rabbit immune serum against S. ja ponicum, indicating its immuno-reactivity. It is concluded that the S. japonicum WD101 protein was predicated as the WD-family protein and it is potential in the signal transduction pathway of schisosomiasis.

关 键 词：日本血吸虫 WD40结构域 生物信息学 克隆 表达 

分 类 号：R383.2[医药卫生—医学寄生虫学]